| Summary: | Hepatitis C virus (HCY) causes a chronic infection that afflicts approximately 2.2% of the world's population. It has a positive-sense RNA genome that encodes a single open reading frame,· translated as a single polyprotein, coding structural proteins at the amino-terminus and non-structural proteins at the carboxyl-terminus. Processing at the NS2INS3 junction of the hepatitis C virus polyprotein occurs via an autoprotease comprising the cytoplasmic region of NS2 and the protease domain of NS3. Mutational analysis has been used to demonstrate that the cysteine residues required for zinc co-ordination in NS3 are similarly required for NS2/3 autoprocessing and to identify a new critical residue - cysteine 922. Detailed examination of zinc coordination in NS3 and NS2/3 using zinc chelation also revealed structural information about NS3 in the context of NS2/3 autoprotease activity. Until recently HCY could not be cultured in cells, therefore subgenomic replicons were developed as a tool to study HCY replication and the behaviour of non-structural proteins in a cellular environment. These replicons were originally produced beginning at either NS2 or NS3, those lacking NS2 were found to replicate more efficiently and were used in almost all subsequent studies. In this work replicons beginning at NS2 were produced with additional upstream sequences to examine their role in the behaviour of NS2 and replication. Some HCY-derived upstream sequences had a lethal effect on replicon replication when present in an atypical context, while an upstream sequence present in a non-lethal context regulates both NS2 localisation and the abundance of non-structural proteins.
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